Useful Pyrex glass etching method using HF and H 2SO 4 mixed acid was studied. To realize the etching behaviors of constant etch rate, high masks durability, small under cut and pinhole free, optimum composition of HF-H 2SO 4 system was found out. Pinhole-free etching, constant etching rate of 0.5 μm/min and smooth surface of less than 4.6nm was obtained at 0.8vol% HF and 32vol% H 2SO 4. This system realized high durability of the etching mask of Si, Cr/Au and even photoresists. This method is useful not only to fabricate Pyrex glass microchannels but also to make PDMS molds. We fabricated a prototype of the pneumatic actuated microvalve system for the single molecular imaging under Total Internal Reflection Fluorescence Microscopy (TIRFM). The leakage free valve actions are confirmed and the switching time of Open-Close and Close-Open modes are 100msec and 120msec respectively.